Notch Signaling Regulates NLRP3 Inflammasome Activation In Acetaminophen-Induced Liver Injury

Objective:An overdose of acetaminophen(N-acetyl-p-aminophenol,APAP)is the most common cause of severe liver damage,ultimately leading to acute liver failure(ALF).The mouse models of APAP-induced liver injury and clinical translational studies revealed that APAP-induced liver damage is not only influenced by the dose of APAP and the initial hepatocyte injury but also by the inflammatory response,which is recognized by activating hepatic macrophages(Kupffer cells)and neutrophils.This activation leads to the release of pro-inflammatory mediators.Notch signaling is highly conserved and critically involved in cell growth,differentiation,and survival.In the immune system,Notch signaling controls the homeostasis of several innate cell populations and regulates immune cell development and function.Activation of Notchl and its ligand Jagged-1 increases cell growth and differentiation during liver regeneration.Disruption of Notch signaling increases intracellular ROS production and inflammatory response,leading to aggravated liver injury induced by ischemia and reperfusion.The NLRP3(nucleotide-binding domain leucine-rich repeat containing family,pyrin domain containing 3)inflammasome is a member of the NLR family of cytosolic pattern recognition receptors and can be activated by various pathogen-associated molecular patterns(PAMPs)and danger-associated molecular patterns(DAMPs)released from stressed or injured cells.NLRP3 regulates the innate immune system through activation of caspase-1 to induce the maturation and secretion of IL-1?.Accumulated evidence demonstrates the important role of NLRP3 in the regulation of immune response associated with inflammatory diseases.We previously found that disruption of NLRP3 inhibited caspase-1 activity and IL-1? production,resulting in reduced liver inflammation in ischemia and reperfusion injury.However,little is known about how Notch signaling may regulate NLRP3 activation during APAP-induced liver injury.In the present study,we identified the novel regulatory role of Notch signaling in APAP-induced liver injury.We demonstrated that blocking Notch pathway triggers NLRP3 inflammasome activation through activation of HMGB1/TLR4/NF-?B signaling,which leads to augmented inflammatory responses and aggravated APAP-induced liver damage.Methods:1.Using a mouse model of APAP-induced liver injury,wild-type(WT)and toll-like receptor 4 knockout(TLR4 KO)mice were injected intraperitoneally with APAP or PBS.2.Some animals were injected with y-secretase inhibitor DAPT or DMSO vehicle.3.For the in vitro study,bone marrow-derived macrophages(BMMs)were transfected with Notchl siRNA,TLR4 siRNA,and non-specific(NS)siRNA and stimulated with LPS.Results:1.Blockade of Notch signaling pathway aggravates APAP-induced liver damage Mice treated with DAPT showed increased sALT levels compared to the DMSO-treated controls at 24 hours after APAP challenge.In contrast,mice treated with vehicle DMSO controls showed less hepatic hemorrhage and necroinflammation after APAP challenge.Livers in mice revealed more hepatic hemorrhage and severe necrosis after receiving DAPT.The mortality was dramatically increased in DAPT-treated mice compared to vehicle DMSO-treated controls.2.Blockade of Notch signaling pathway augments macrophage/neutrophil trafficking in APAP-induced liver injuryIndeed,DAPT treatment augmented CD68+ macrophage infiltration compared to the DMSO controls at 24 hours after APAP challenge.Moreover,the MPO assay was increased in livers of mice subjected to DAPT condition(5.31±0.12)compared to the DMSO(3.27±0.21,p<0.05)or WT controls(3.05±0.13,p<0.05).3.Blockade of Notch signaling pathway inhibits Hesl and promotes HMGB1/TLR4/NF-?B and NLRP3 activation in APAP-induced liver injuryBy 24 hours of APAP challenge,the protein expression of Hesl was down-regulated in DAPT-but not in DMSO-treated livers.However,DAPT treatment up-regulated the protein expression of HMGB1,TLR4,NF-?B,and NLRP3 in APAP-challenged livers.Moreover,livers in mice receiving DAPT increased the mRNA levels of proinflammatory genes coding for IL-1?,TNF-?,and IL-6 compared to the DMSO controls.4.Blockade of Notch signaling pathway promotes APAP-induced hepatocellular apoptosisIndeed,DAPT treatment inhibited phosphorylation of Stat3 and Akt compared to the DMSO controls.The increased expression of cleaved caspase-3 was observed in DAPT-but not in DMSO-treated livers.This result was further confirmed by TUNEL staining.We found that DAPT preconditioning augmented the frequency of TUNEL+cells in APAP-challenged liver lobes(44.5±1.4),as compared with DMSO(24.2±0.9,p<0.001)or WT(22.2±.7,p<0.001)controls.5.Disruption of TLR4 signaling ameliorates APAP-induced liver damage and inhibits NLRP3 activation.At 24 hours of DAPT precondition,livers in WT mice showed severe necrosis.In contrast,livers in TLR4 KO mice after receiving DAPT revealed mild hemorrhage without necrosis.DAPT treatment increased the expression of NF-?B,NLRP3,and cleaved caspase-1 in APAP-challenged mice.However,disruption of TLR4 in DAPT-treated mice resulted in reduced NF-?B,NLRP3,and cleaved caspase-1.The expression of pro-inflammatory IL-1?,TNF-?,and IL-6 consistently decreased in TLR4 KO but not in WT mice treated with DAPT.6.Notch signaling regulates HMGB1/TLR4/NF-?B and NLRP3 activation in vitroUnlike in the control NS siRNA group,knockdown of Notchl with Notchl siRNA pretreatment depressed Hesl expression and phosphorylated Stat3 and Akt in LPS-stimulated BMMs.However,increased HMGB1,TLR4,NF-?B,and NLRP3 expression was observed in Notchl siRNA-but not in the NS siRNA-treated group.Moreover,Notchl treatment augmented the mRNA levels of IL-1p,TNF-a,and IL-6 in BMMs after LPS stimulation compared to the NS siRNA-treated controls.7.TLR4 signaling is essential for Notch-mediated immune regulation of NLRP3 activation in vitroDAPT precondition increased the expression of NF-?B,NLRP3 and cleaved caspase-1 in NS siRNA-transfected BMMs.However,knockdown of TLR4 by TLR4 siRNA transfection diminished NF-?B,NLRP3,and cleaved caspase-1 in DAPT-treated cells.Consistent with this data,knockdown of TLR4 resulted in reduced mRNA levels coding for IL-1?,TNF-?,and IL-6 in LPS-stimulated BMMs after DAPT treatment.These results imply that TLR4 signaling might be essential for the Notch-mediated immune regulation of NLRP3 activation during inflammatory response.Conclusion:1.Blockade of Notch signaling aggravates APAP-induced liver damage.2.Blockade of Notch signaling augments macrophage/neutrophil trafficking,and induces hepatocellular apoptosis.3.Blockade of Notch signaling promotes HMGB1/TLR4/NF-?B and NLRP3 activation via a TLR4-dependent pathway.Thus,these findings document the key mechanism of Notch signaling in regulating innate immune responses in APAP-induced liver injury.